The development of real-time methods for the measurement of blood drug levels would revolutionize many aspects of contemporary healthcare. It could, for example, provide a means of regulating drug delivery (e.g....chemotherapeutics) on-the-fly which, in turn, could greatly improve drug efficacy while reducing the potentially toxic consequences of over-dosage. The goal of the proposed research is to adapt an existing, electrochemical aptamer-based (E-AB) sensing technology to meet this demanding application. Critical to this approach, the E-AB platform has already proven sensitive (micromolar) reusable, rapid (seconds - minutes), reagentless, and selective enough to employ directly in blood serum and other complex, clinically relevant sample matrices. However, while E-AB sensors perform well when challenged with urine, blood serum and other complex materials under ideal laboratory conditions, the technology requires further development before it will achieve clinically-relevant detection in flowing, whole blood. Thus motivated, I hereby propose the development of methods to improve the sensitivity, stability and detection confidence of the E-AB platform in order to improve this technology such that it meets the demands of this ambitious goal. To do so I will focus on three specific aims: 1) optimize sensor detection limits and response times via a systematic study of the effect of aptamer biophysics and sensor fabrication on E-AB signaling, 2) improve sensor performance for operation in whole blood via the synthesis and application of alternative redox tags, and 3) develop methods to measure and correct E-AB background current thus improving detection accuracy and confidence. Contained within each of these specific aims are multiple, complimentary strategies for characterization, modification and optimization of the biophysical properties of DMA aptamer sensors that should significantly improve our understanding of this potentially important new diagnostic technology. The development of real-time methods for monitoring drug levels, such as therapeutics, in flowing blood would revolutionize modern healthcare at the point-of-care. Point-of-care detection could, for example, enable feedback controlled drug dosage of unprecedented precision that is individualized to the patient. Here I propose the development of sensors, with the ultimate, if ambitious, goal of being capable of supporting the real-time quantification of drugs directly in whole blood.